Platinum complex and anti-tumor agent comprising said complex as active ingredient

ABSTRACT

Disclosed are a novel platinum complex represented by the following formula I or a steric isomer thereof, an anti-tumor agent comprising this complex as the effective ingredient, and a method of curing a tumor by using this complex: ##STR1## This complex is advantageous in that the toxicity is low and the anti-tumor activity is high.

TECHNICAL FIELD

The present invention relates to a novel platinum complex having ananti-tumor activity and which is valuable as a medicine such as ananti-tumor agent.

BACKGROUND ART

Some platinum complexes represented by cisplatin have a remarkableanti-tumor effect, and cisplatin has been applied to various cases.Nevertheless, the toxicity, such as the kidney toxicity, of cysplatin isvery strong, and this strong toxicity is an obstacle to a medicaltreatment. Accordingly, the development of a medicine having a toxicitylower than those of conventional platinum complexes, and a higheranti-tumor effect, is required.

DISCLOSURE OF THE INVENTION

With a view to solving this problem, the present inventors synthesizedvarious platinum complexes and examined the anti-tumor effects thereof,and as the result, the inventors found a platinum complex having atoxicity lower than those of the conventional platinum complexes, and ahigher anti-tumor activity, and the present invention was completedbased on this finding.

More specifically, in accordance with the present invention, there areprovided a novel platinum complex represented by the following formula Ior a steric isomer thereof (hereinafter referred to as "the compound ofthe present invention"): ##STR2## and an anti-tumor agent comprisingthis complex as an active ingredient.

Furthermore, there is provided a method of remedying a tumor with thecompound of the present invention.

BEST MODE OF CARRYING OUT THE INVENTION

The steric isomer of the compound of the formula I includes compoundsrepresented by the following formulae ##STR3##

The compound of the present invention can be prepared, for example,according to the following procedures.

A dinitrato platinum complex having 1,2-cyclooctanediamine as a carrierligand is used as the starting material, and this complex is broughtinto contact with an OH⁻ anion exchange resin, to convert the nitrogroup of the complex to a hydroxyl group. The intended complex isobtained by reacting the above complex with oxalic acid, andrecrystallization can be carried out by using an appropriate solventsuch as water, according to need.

Furthermore, after the termination of the reaction, a filtration can becarried out by using an adsorbent such as active carbon, according toneed, whereby a more refined crystal can be obtained.

As a specific example of the dinitrato platinum complex, there can bementioned dinitrato(diaminocyclooctane)platinum (II).

As the dinitrato(diaminocyclooctane)platinum (II), there are knowndinitrato-(1R,2R)-diaminocyclooctane platinum (II) anddinitrato-(1S,2S)-diaminocyclooctane platinum (II) as the trans-form,and dinitrato-cis-diaminocyclooctane platinum (II) as the cis-form. Thepresent invention includes all of the corresponding isomers preparedfrom these starting materials and mixtures comprising these isomers atoptional ratios.

As a specific example of the OH⁻ type anion exchange resin, there can bementioned Diaion SA10AOH (supplied by Mitsubishi Kasei).

As the contact method, any method whereby the platinum complex isbrought into contact with the resin, for example, the batch methods andcolumn methods, can be adopted, but in view of the operation efficiency,a column method is preferably adopted.

For the reaction with oxalic acid, there is preferably adopted a methodin which oxalic acid dihydrate is dissolved in an amount slightlysmaller than the chemical equivalent to the starting platinum complex,and the solution is allowed to stand for about 12 to about 36 hours.After termination of the reaction, the aqueous solution is concentratedor a precipitation is effected by addition of a solvent such asmethanol, and the precipitated crystal is recovered by filtration anddried to obtain the intended complex.

The structure of the compound of the present invention has beenconfirmed by elementary analysis, infrared absorption spectrum analysisand the like.

The anti-tumor effect of the compound of the present invention will nowbe described with reference to the following experiments.

Experiment 1

L1210 mouse leukemia cells, P388 mouse leukemia cells, Hela humanuterine neck cancer cells, T24 human bladder cancer cells or MCF7 humanmammary cancer cells were suspended in Eagle medium (Hela and T24)containing 10% bovine fetal serum, PRMI1640 medium (L1210 and MCF7) orPRM11740 medium (P388) mixed with 5×10⁻⁵ M mercaptoethanol, and thecells were seeded on a 96-well plate at an inoculum size of 3×10³ cellsper hole. A compound obtained in an example described below or cysplatin(CDDP) was diluted with physiological saline solution and added to themedium at various concentrations. Culturing was conducted at 37° C. for48 hours in a carbon dioxide incubator, and the propagation of cells wasdetermined by the MTT [3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide] method (J. Immunol. Method, 65, 55, 1983). Theabsorbance A after 48 hours of culturing at each concentration, theabsorbance B after 48 hours of culturing in the control, and theabsorbance C before addition of the compound obtained in the examplewere determined, and the propagation reaction rate D of eachconcentration was calculated from the following formula: ##EQU1## Theconcentration IC₅₀, unit: μg/ml) of the compound obtained in the examplegiving the value corresponding to 50% of the value of the control wasdetermined from the curve showing the relationship between theconcentration of the compound obtained in the example and thepropagation reaction rate.

The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Inhibition of Propagation of Mouse and Human Cancer Cells                             IC.sub.50 (μg/ml)                                                  Cell Name CDDP    Compound Obtained in Example 1                              ______________________________________                                        L1210     0.46    0.03                                                        P388      0.09    0.06                                                        P388      0.09    0.06                                                        HeLa      1.42    0.29                                                        T24       1.51    1.43                                                        MCF7      3.28    0.41                                                        ______________________________________                                    

Experiment 2

Mouse leukemia L1210 cells (1×10⁵) were transplanted into the abdominalcavity of a 6-weeks-old female BDF₁ mouse. Then, continuously for 3 daysfrom the next day, a compound of a compound obtained in an exampledescribed below or CDDP was administered to the abdominal cavity once aday. In the control group, physiological saline solution alone wassimilarly administered.

The anti-tumor effect was evaluated based on the life prolongation ratio(T/C value) determined from the average survival day numbers of thegroup of the compound obtained in the example and the control groupafter 30 days from the transplantation of L1210. On the 30th day fromthe transplantation, the experiment was concluded. In the case of micesurviving for more than 30 days, the survival day number was regarded as30. ##EQU2## The average survival day number at each dose, the T/C value(%) and the ratio of the number of surviving mice upon termination ofthe experiment to the number of tested mice are shown in Table 2. TheILS₃₀ value (the dose giving a 30% life-prolonging effect over thecontrol group), the ILS_(max) value (the dose giving a highestlife-prolonging effect), the TR value (cure ratio; ILS_(max) /ILS₃₀) andthe CR value (the ratio of the number of mice completely cured at theILS_(max) value and the number of mice showing no propagation of a tumorafter 30 days from the transplantation to the number of tested mice) areshown in Table 3.

Note, 1 indicates the ratio of the number of surviving mice to thenumber of tested mice.

                  TABLE 2                                                         ______________________________________                                                  Dose   Average Survival                                                                           T/C                                                       (mg/kg)                                                                              Day Number   (%)     1                                       ______________________________________                                        Compound Obtained                                                                         0.3      >16.8        >183  1/6                                   in Example 1                                                                  Compound Obtained                                                                         1.0      >19.7        >214  1/6                                   in Example 1                                                                  Compound Obtained                                                                         3.0      >20.3        >220  2/6                                   in Example 1                                                                  Compound Obtained                                                                         10.0     >30.0        >326  6/6                                   in Example 1                                                                  ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                   ILS.sub.30                                                                            ILS.sub.max                                                           (mg/kg) (mg/kg)   TR      CR                                       ______________________________________                                        Compound Obtained                                                                          0.06      10.0      167   6/6                                    in Example 1                                                                  CDDP         1.5       6.0        4    3/6                                    ______________________________________                                    

From the foregoing results, it has been confirmed that the compound ofthe present invention has an excellent anti-tumor activity.

When the acute toxicity test of the compound of the present inventionwas carried out by using male mice of the ICR family, it was found thatLD₅₀ of the compound of the present invention was 29.5 mg/kg in the caseof an administration into the abdominal cavity, and since this LD₅₀value of CDDP is 14.6 mg/kg, it is understood that the compound of thepresent invention is safe.

Namely, it is considered that the compound of the present invention hasan excellent anti-tumor activity and is valuable as an anti-tumor agenthaving a low toxicity.

The doses and preparations of the compound of the present invention willnow be described.

The compound of the present invention can be administered to humans andanimals directly or together with a conventional drug carrier. Theadministration mode is not particularly critical, and an appropriateadministration mode is selected according to need. For example, therecan be mentioned an agent for oral administration, such as a tablet, acapsule, a granule, a fine particle or a powder, and an agent fornon-oral administration, such as an injection or a suppository.

In order for the compound of the present invention to attain theintended effect as an agent for oral administration, it is consideredpreferable to administer 10 mg to 1 g of the compound of the presentinvention to an adult per day dividedly in several times, though anappropriate dose is changed according to the age, body weight anddisease state of a patient.

The agent for oral administration can be prepared according to customaryprocedures by using, for example, starch, lactose, refined sugar,mannitol, carboxymethyl cellulose, corn starch and inorganic salts.

The preparation of this type can further comprise a binder, adisintegrating agent, a surface active agent, a lubricant, a flowabilityimprover, a taste improver, a coloring agent, a perfume and the like.Specific examples of these additives are described below.

(Binder)

Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch,methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylcellulose, crystalline cellulose, ethyl cellulose, polyvinyl pyrrolidoneand macrogol.

(Disintegrating Agent)

Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, carboxymethyl cellulose and lowly substitutedhydroxypropyl cellulose.

(Surface Active Agent)

Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester andpolysorbate 80.

(Lubricant)

Talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester,magnesium stearate, calcium stearate, aluminum stearate and polyethyleneglycol.

(Flowability Improver)

Soft silicic anhydride, dry aluminum hydroxide gel, synthetic aluminumsilicate and magnesium silicate.

The compound of the present invention can be administered in the form ofa suspension, emulsion, syrup or elixir. The preparation of this typecan further comprise a taste improver, an odor improver and a coloringagent.

In order for the compound of the present invention to exert the intendedeffect as the agent for non-oral administration, it is consideredpreferable to administer the compound of the present invention in anamount of 5 to 600 mg per day to an adult by intravenous injection,instillation, hypodermic injection or intramuscular injection, thoughthe amount administered is changed according to the age, body weight anddisease degree of a patient.

The agent for non-oral administration can be prepared according to knownprocedures, and as the diluent, there can be used distilled water forinjection, physiological saline solution, an aqueous solution ofglucose, a vegetable oil for injection, sesame oil, peanut oil, soybeanoil, corn oil, propylene glycol and polyethylene glycol. A fungicide, anantiseptic agent and a stabilizer can be added according to need. Inview of the stability of the agent for non-oral administration, a methodcan be adopted in which the agent is filled in a vial or the like, wateris removed by a usual freeze-drying technique and a liquid agent isprepared again just before the administration. Appropriate additivessuch as an isotonic agent, a stabilizer, an antiseptic agent and alenitive agent can be added according to need.

As the other agent for non-oral administration, there can be mentionedcoating agents such as a lotion for external application and anointment, and a suppository for intrarectal application, and these canbe prepared according to customary procedures.

The present invention will now be described in detail with reference tothe following examples, that by no means limit the scope of theinvention.

EXAMPLE 1

Dinitro-trans-cyclooctanediamine-platinum as the starting material wassynthesized by using transcyclooctanediamine containing optical isomershaving (1R,2R) and (1S,2S) arrangements at a ratio of 1:1 as the carrierligand. Then, 12.4 g of this trans-compound was dissolved in 400 ml ofdistilled water, and the solution was passed through a column filledwith 300 ml of Diaion SA10AOH and fluxed with distilled water. Then, 3.3g of oxalic acid was dissolved in the effluent and reaction was carriedout at 30° C. for 12 hours. After termination of the reaction, thereaction liquid was passed through a column filled with active carbon asthe adsorbent and fluxed with distilled water. The effluent wasconcentrated, and recrystallization was carried out by using water toobtain 8.5 g of a light yellow crystal.

By the following physical and chemical properties, this yellow crystalwas identified as a 1:1 mixture of oxalate-(1R,2R)-diaminocyclooctaneplatinum (II) and oxalate-(1S,2S)-diaminocyclooctane platinum (II),included in the scope of the compound of the present invention.

Elementary Analysis Values (as C₁₀ H₁₈ O₄ N₂ Pt)

Calculated values (%): C=28.23, H=4.27, N=6.59

Measured values (%): C=28.26, H=4.25, N=6.65

Infrared Absorption Spectrum ν_(max) ^(KBr) cm⁻¹ 3450, 3050, 1710, 1390,815

EXAMPLE 2

In methanol was dissolved 10 mg of the compound obtained in Example 1,and the solution was subjected to optical resolution by columnchromatography using a separating optical resolution column [UltronES-OVM (20 mm in diameter and 25 cm in length; particle size of 10 μm;supplied by Shinwa Kako).

The column conditions were such that the moving phase comprised 100 mMsodium perchlorate (0.2% H₃ PO₄) and acetonitrile at a ratio of 90:10,the flow rate was 0.5 ml/min, the column temperature was maintained at aconstant level of about 25° C., and detection was carried out by RI(RANGE 1/2). The fraction A flowing out in the vicinity of 4 minutes and30 seconds and the fraction B flowing out in the vicinity of 6 minutesand 30 seconds were collected. Each fraction was subjected to adesalting operation and the solvent was removed by distillation, whereby4.5 mg of (-)-oxalate diaminocyclooctane platinum (II) having a specificrotation described below and 5.0 mg of (+)-oxalate diaminocyclooctaneplatinum (II) having a specific rotation described below were obtained.

Specific Rotations

Fraction A: [α]_(D) ²⁰ =-55° (C=0.1, MeOH), Fraction B: [α]_(D) ²⁰ =+46°(C=0.1, MeOH)

EXAMPLE 3

    ______________________________________                                        (1) Corn starch          52     g                                             (2) Crystalline cellulose                                                                              40     g                                             (3) Calcium carboxymethyl cellulose                                                                    5      g                                             (4) Light silicic anhydride                                                                            0.5    g                                             (5) Magnesium stearate   0.5    g                                             (6) Compound obtained in Example 1                                                                     2      g                                             Total                    100    g                                             ______________________________________                                    

According to the above-mentioned recipe, components (1) through (6) werehomogeneously mixed, and the mixture was compression-molded by atableting machine to obtain tablets, each having a weight of 200 mg.

Each tablet contained 4 mg of the compound obtained in Example 1, Threeto 50 tablets were administered to an adult per day, dividedly severaltimes.

EXAMPLE 4

    ______________________________________                                        (1) Crystalline cellulose                                                                              92.5   g                                             (2) Magnesium stearate   0.5    g                                             (3) Calcium carboxymethyl cellulose                                                                    5      g                                             (4) Compound obtained in Example 1                                                                     2      g                                             Total                    100    g                                             ______________________________________                                    

According to the above-mentioned recipe, components (1) and (4) and apart of component (2) were homogeneously mixed, the mixture wascompression-molded and the molded product was pulverized. Component (3)and the remainder of component (2) were mixed with the pulverizedproduct, and the mixture was compression-molded by a tableting machineto obtain tablets, each having a weight of 200 mg. Each tablet contained4 mg of the compound obtained in Example 1. Three to 50 tablets wereadministered to an adult per day, dividedly several times.

EXAMPLE 5

    ______________________________________                                        (1)    Crystalline cellulose  42.5   g                                        (2)    10% Solution of hydroxpropyl                                                                         50     g                                               cellulose in ethanol                                                   (3)    Calcium carboxymethyl cellulose                                                                      5      g                                        (4)    Magnesium stearate     0.5    g                                        (5)    Compound obtained in Example 1                                                                       2      g                                               Total                  100    g                                        ______________________________________                                    

According to the above-mentioned recipe, components (1), (2) and (5)were homogeneously mixed and kneaded according to customary procedures,and the mixture was granulated by an extrusion granulator and thegranulation product was dried and disintegrated. Then, thedisintegration product was mixed with components (3) and (4) and themixture was compression-molded by a tableting machine to obtain tablets,each having a weight of 200 mg.

Each tablet contained 4 mg of the compound obtained in Example 1. Threeto 50 tablets were administered to an adult per day, dividedly severaltimes.

EXAMPLE 6

    ______________________________________                                        (1) Corn starch          93     g                                             (2) Magnesium stearate   0.5    g                                             (3) Calcium carboxymethyl cellulose                                                                    5      g                                             (4) Light silicic anhydride                                                                            0.5    g                                             (5) Compound obtained in Example 1                                                                     1      g                                             Total                    100    g                                             ______________________________________                                    

According to the above-mentioned recipe, components (1) through (5) werehomogeneously mixed, compression-molded by a compression moldingmachine, pulverized by a pulverizing machine and sieved to obtain agranule.

In 1 g of this granule was contained 10 mg of the compound obtained inExample 1, and 1 to 20 g of the granule was administered to an adult perday, dividedly several times.

EXAMPLE 7

    ______________________________________                                        (1)    Crystalline cellulose  69     g                                        (2)    10% Solution of hydroxypropyl                                                                        30     g                                               cellulose in ethanol                                                   (3)    Compound obtained in Example 1                                                                       1      g                                               Total                  100    g                                        ______________________________________                                    

According to the above-mentioned recipe, components (1) through (3) werehomogeneously mixed and kneaded, granulated by an extrusion granulator,dried, and sieved to obtain a granule.

In 1 g of this granule was contained 10 mg of the compound obtained inExample 1, and 1 to 20 g of the granule was administered to an adult perday, dividedly several times.

EXAMPLE 8

    ______________________________________                                        (1) Corn starch          97.5   g                                             (2) Light silicic anhydride                                                                            0.5    g                                             (3) Compound obtained in Example 1                                                                     2      g                                             Total                    100    g                                             ______________________________________                                    

According to the above-mentioned recipe, components (1) through (3) werehomogeneously mixed, and 200 mg of the mixture was filled in a capsuleNo. 2.

Each capsule contained 4 mg of the compound obtained in Example 1, and 3to 50 capsules were administered to an adult per day, dividedly severaltimes.

EXAMPLE 9

    ______________________________________                                        (1)    Distilled water for                                                                          appropriate amount                                             injection                                                              (2)    Glucose        200        mg                                           (3)    Compound obtained in                                                                         100        mg                                                  Example 1                                                                     Total          5          ml                                           ______________________________________                                    

Components (2) and (3) were dissolved in distilled water for injection,and the solution was charged into an ampoule having a capacity of 5 mland was sterilized at 121° C. for 15 minutes under pressure to obtain aninjection.

INDUSTRIAL APPLICABILITY

The present invention can be effectively used in the medicinal field asa remedy for tumors.

We claim:
 1. A novel platinum complex represented by the followingformula I or a steric isomer thereof: ##STR4##
 2. An anti-tumor agentcomprising as the active ingredient a platinum complex represented bythe following formula I or a steric isomer thereof: ##STR5##
 3. A methodof curing tumors, which comprises using a platinum complex representedby the following formula I or a steric isomer thereof: ##STR6##